Induction of cell death of virus-infected cells limits the viral factory and facilitates cross priming of virus- specific T cells. As such, cell death is an important host response against viral pathogens. In response, viruses often encode inhibitors that counteract host cell death. For example, many herpesviruses and poxviruses encode inhibitors of caspases, the key enzymes that execute apoptosis. In many cases, these viral inhibitors can dramatically alter the host response against the virus. Necroptosis or programmed necrosis is a non-apoptotic form of cell death marked by rapid loss of plasma membrane integrity that often precedes exposure of the ?eat-me? signal phosphatidyl serine (PS) on the cell surface. Receptor interacting protein kinase 3 (RIPK3) is the critical kinase that mediates necroptosis. RIPK3 phosphorylates its downstream effector mixed lineage kinase domain-like (MLKL). Phospho-MLKL undergoes oligomerization, translocates to the plasma membrane and induces membrane rupture. The release of cellular damage-associated molecular patterns (DAMPs) from necroptotic cells stimulates innate immune effectors such as dendritic cells and macrophages to enhance anti-viral immune responses. Because RIPK3 and its upstream activator RIPK1 are both cleavage substrates of caspase 8, optimal induction of necroptosis requires inhibition of caspases. Previously, we found that cells infected with vaccinia virus (VV), the poxvirus strain that was used as vaccine against smallpox, were highly sensitive to TNF-induced necroptosis. Ripk3-deficient mice failed to control VV replication and succumbed to the infection. These observations led to the current dogma that poxviruses sensitize infected cells to necroptosis. In contrast to VV, several recent reports showed that herpesviruses often encode viral inhibitors of necroptosis. These results led us to ask whether VV-induced sensitization to necroptosis is a universal phenomenon for poxviruses. In preliminary studies, we found that unlike VV-infected cells, cowpox virus (CPXV) infected cells were resistant to TNF-induced necroptosis. CPXV induces resistance to necroptosis by proteasome-mediated degradation of the essential signal adaptor RIPK3. Moreover, we found that the viral caspase 8 inhibitor MC159 from the human poxvirus Molluscum contagiosum virus (MCV) is a dual function inhibitor that inhibits apoptosis and necroptosis. Based on these preliminary results, we propose two aims to investigate the molecular mechanisms by which CPXV and MC159 inhibit TNF-induced necroptosis. In Aim 1, we will screen and identify the CPXV gene product that mediates proteasome-mediated RIPK3 degradation. Specifically, we will test the hypothesis that the E3 ligase CPXV-p28 is the key viral enzyme that mediates this effect. We will use RNA interference and generate recombinant VV expressing either wild type or mutant p28 to determine the functional role of RIPK3 degradation on viral replication. In Aim 2, we will interrogate the molecular mechanism by which MC159 inhibits necroptosis. Specifically, we will test the hypothesis that MC159 binds to the cellular E3 ligases cIAP1 and cIAP2 to inhibit their recruitment to the TNFR1 signaling complex. Together, these studies will yield important knowledge on the viral mechanisms of cell death inhibition and the role of necroptosis in virus evolution.